2023 Volume 48 Issue 2 Pages 65-73
Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces various biological reactions in vivo. Our previous study suggested that LPS administration disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in the liver mitochondria, and sensitizes mitochondrial permeability transition (MPT) pore opening in rats. However, it is unknown whether LPS-induced MPT pore opening in rats is similarly observed in mice and whether the mechanism is the same. LPS administration to mice increased not only cyclosporin A-sensitive swelling (MPT pore opening) susceptibility, but also induced cyclosporin A-insensitive basal swelling, unlike in rats. In addition, respiratory activity observed after adding ADP was significantly decreased. Based on these results, we further investigated the role of adenine nucleotide translocase (ANT). Carboxyatractyloside (CATR; an ANT inhibitor) treatment decreased respiratory activity after ADP was added in vehicle-treated mitochondria similarly to LPS administration. Additionally, CATR treatment increased MPT pore opening susceptibility in LPS-treated mitochondria compared to that of vehicle-treated mitochondria. Our study shows that ANT maintained a c-state conformation upon LPS administration, which increased MPT pore opening susceptibility in mice. These results suggest that LPS enhances MPT pore opening susceptibility across species, but the mechanism may differ between rat and mouse.
Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is an inflammatory mediator that activates several immune cells via Toll-like receptor-4 (TLR4) (Alexander and Rietschel, 2001). LPS is often used in basic research; in particular, administration of high concentrations is especially useful for the purposes of generating murine sepsis models (Dickson and Lehmann, 2019). In an LPS-induced sepsis model, the liver is one of the major target organs. Previous reports have suggested that LPS administration leads to liver injury by activation of Kupffer cells (Su, 2002) and that the development of LPS-induced liver injury may be due to the opening of the mitochondrial permeability transition (MPT) pore via the generation of oxidative stress (Zhuge and Cederbaum, 2009; Veres et al., 2021).
The MPT is a phenomenon characterized by increased inner membrane permeability. It is thought to evoke cell death via the release of cytochrome c into the cytosol or via a decrease in ATP levels (Hurst et al., 2017). MPT pore opening leads to ischemia and reperfusion injury (in the brain (Li et al., 2018), heart (Zhang et al., 2019), and lungs (Tian et al., 2018)) as well as severe drug-induced liver injury (Labbe et al., 2008). There have been decades-long discussions regarding the structure of the MPT pore. Recently, it is proposed that ATP synthasomes, including ATP synthase, adenine nucleotide translocase (ANT), and inorganic phosphate carriers in the inner membrane, are major components of MPT pores (Bonora et al., 2022). In addition, two models of MPT pore conductance have been proposed (Bonora et al., 2022). ATP synthase forms high-conductance MPT pore under conditions of high stress. Thus, pore opening affects mitochondrial function and leads to cell death. In contrast, ANT forms low-conductance MPT pore under normal physiological condition. The mechanism of MPT pore opening involves Ca2+ accumulation in the mitochondrial matrix and the interaction of cyclophilin D and ATP synthase (Giorgio et al., 2018, 2010). Additionally, oxidative stress, inorganic phosphate, and decreased mitochondrial membrane potential also induce MPT pore opening (Morciano et al., 2021).
Our previous study suggested that LPS administration disrupts respiratory chain complex activities, enhances reactive oxygen species production, particularly in the mitochondria, and sensitizes MPT pore opening in rats (Arakawa et al., 2019). It has also been established that LPS administration induces MPT pore opening in mice (Veres et al., 2021). However, there is a significant difference in the immune cell response to LPS administration between rats and mice. In mice, several macrophage-related genes are upregulated by LPS treatment, however these genes remain constantly expressed and unaltered in rats (Pridans et al., 2021). Generally, an intracellular energy shift is triggered during macrophage differentiation; a recent study has suggested that the involvement of interleukin-10 is critical for the differentiation of activated macrophages following LPS treatment (Dowling et al., 2021). Based on these reports, various inflammatory conditions caused by LPS administration can have different effects on mitochondrial function.
Our previous study (Arakawa et al., 2019) was limited to isolated rat liver mitochondria and it is still unknown whether MPT pore opening susceptibility is increased by LPS treatment in isolated mouse liver mitochondria. Therefore, the present study is the first to confirm that LPS administration leads to increased MPT pore opening susceptibility in mouse livers under specific experimental conditions. Based on these results, we also hypothesized that LPS administration would affect ANT function, especially maintaining ANT’s conformation. Finally, we evaluated ANT function using a specific ANT inhibitor and attempted to elucidate the mechanism of increased MPT pore opening susceptibility in isolated liver mitochondria from LPS-treated mouse.
LPS from Escherichia coli O127:B8 (lot number: 0000082455, 1.5 × 106 endotoxin units/mg), perchloric acid, thiobarbituric acid, antimycin A, decylubiquinone, rotenone, dichloroindophenol, oligomycin, and rhodamine 123 were purchased from Millipore-Sigma (St. Louis, MO, USA). Cytochrome c, 1,1,3,3-tetraethoxypropane, cyclosporin A, collagenase, dexamethasone, and insulin were purchased from FUJIFILM Wako Pure Chemical Industries Ltd. (Osaka, Japan). ADP, ATP, pyruvate kinase, and lactate dehydrogenase were purchased from Oriental Yeast (Tokyo, Japan). Carboxyatractyloside and bongkrekic acid were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). William’s medium E (WME) and GlutaMAXTM were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Biosera (Nuaille, France). Antibiotic-antimycotic solutions were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Type I collagen (rat tail) was purchased from Corning (Bedford, MA, USA).
AnimalsAll animal studies were conducted following the Principles of Laboratory Animal Care, as adopted and promulgated by the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publication No. 8023, revised 1978), USA, and the Guidelines for Animal Studies provided by Chiba University. All protocols were approved by the Institutional Animal Care and Use Committee of Chiba University (number: dou 1–222). C57BL/6J male mice (Charles River Laboratories Japan Inc., Kanagawa, Japan), aged 8–12 weeks, were used in this study. Animals were housed in an air-conditioned room (25°C) under a 12 hr light/dark cycle for at least 1 week before use. Food (FR-1 diet; Oriental Yeast Co., Ltd., Tokyo, Japan) and water were provided ad libitum.
Preparation of mouse liver mitochondriaAfter overnight fasting, the mice were treated with LPS (1 mg/kg) or saline intraperitoneally and simultaneously heparin (3,000 unit/kg) or saline intravenously, and access to food was restored. After 2 hr, the liver was isolated. Liver mitochondria were isolated as previously described (Sato et al., 2021; Arakawa et al., 2019).
Measurement of mitochondrial swellingMitochondrial swelling was evaluated using a previously described method (Arakawa et al., 2019) with some modifications. Mitochondria (0.5 mg/mL) were pre-incubated in swelling buffer (125 mM sucrose, 65 mM KCl, 5 mM succinate, 10 mM HEPES-KOH, 2.5 µM rotenone, pH 7.4) at 30°C. The absorbance at 540 nm was monitored using a UV-2550 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) after adding 12.5 µM Ca2+ (MPT inducer) and 2 μM cyclosporin A (CsA, MPT inhibitor).
Measurement of thiobarbituric acid reactive substances (TBARS) in isolated mitochondria and liver homogenatesLiver homogenates and mitochondrial TBARS were evaluated using a previously described method (Arakawa et al., 2019) with some modifications. Samples were centrifuged at 15,000 × g for 5 min at 4°C, and the supernatant was removed. The pellet was washed twice with cold PBS and mixed with 3.86% perchloric acid and 20 mM thiobarbituric acid. The mixture was heated for 30 min at 100°C and centrifuged at 2,000 × g for 15 min at room temperature. The supernatant was collected and its absorbance was measured at a wavelength 400–600 nm using a UV-2550 spectrophotometer. Malondialdehyde (MDA; lipid peroxidation products) levels were determined by subtracting the absorbance at 570 nm (regarded as the background) from the absorbance at 532 nm.
Measurement of mitochondrial respiratory activity using oxygen consumption rateMitochondrial respiratory activity was evaluated using a previously described method (Arakawa et al., 2019). Dissolved oxygen concentration was measured using an OxoDish® OD24 and SDR SensorDish® Reader (PreSense).
Measurement of respiratory chain complex activityAbsorbance was measured using a UV-2550 spectrophotometer for 6–15 min as follows. Net absorbance was defined by subtracting the absorbance with a specific inhibitor from the absorbance without a specific inhibitor at each time point. After that, net absorbance was plotted, and the slope of a line (regarded as respiratory chain complex activity) was calculated.
Complex IComplex I specific activity was evaluated using a previously described method (Ma et al., 2011) with some modifications. Mitochondria (50 μg/mL) were pre-incubated in complex I buffer (1 mM MgCl2, 30 mM Kpi, 2 mM KCN, 5 μg/mL antimycin A, 0.67% BSA, 70 μM decylubiquinone, pH 7.5) for 3 min at 30°C. After 200 μM NADH was then added, the decrease in absorbance at 340 nm was measured in the absence or presence of 50 μM rotenone (complex I inhibitor).
Complex IIComplex II-specific activity was evaluated using a previously described method (Trounce et al., 1996) with some modifications. Mitochondria (50 μg/mL) were preincubated in complex II buffer (50 mM Kpi, 2 mM KCN, 5 μg/mL antimycin A, 50 μM rotenone, 50 μM decylubiquinone, 50 μM dichloroindophenol, pH 7.5) for 3 min at 30°C. After the addition of 5 mM succinate, the decrease in absorbance at 600 nm was measured in the absence or presence of 10 mM malonate (complex II inhibitor).
Complex IIIComplex III-specific activity was evaluated using a previously described method (Trounce et al., 1996) with some modifications. Mitochondria (25 μg/mL) were preincubated in complex III buffer (1 mM MgCl2, 30 mM Kpi, 2.5% BSA, 2 mM KCN, 50 μM rotenone, 100 μM decylubiquinol, pH 7.5) for 3 min at 30°C. After 25 μM of oxidized cytochrome c was added, the increase in absorbance at 550 nm was measured in the absence or presence of 5 μg/mL antimycin A (complex III inhibitor). Decylubiquinol was produced from decylubiquinone using a previously described method (Spinazzi et al., 2012).
Complex IVComplex IV-specific activity was evaluated using a previously described method (Ma et al., 2011) with some modifications. Mitochondria (3.125 μg/mL) were pre-incubated in 50 mM Kpi, pH 7.5 for 3 min at 30°C. After 20 μM of reduced cytochrome c was added, the decrease in absorbance at 550 nm was measured in the absence or presence of 2 mM KCN (complex IV inhibitor). Reduced cytochrome c was produced from oxidized cytochrome c using a previously described method (Spinazzi et al., 2012).
Complex VComplex V-specific activity was evaluated using a previously described method (Ma et al., 2011) with some modifications. Mitochondria (250 μg/mL) were preincubated in complex V buffer (10 mM HEPES-MgSO4, 300 μM NADH, 2.5 mM phosphoenolpyruvate, 500 μg/mL pyruvate kinase, 500 μg/mL lactate dehydrogenase, 5 μg/mL antimycin A, pH 7.5) for 3 min at 30°C. After 2.5 mM ATP was added, the decrease in absorbance at 340 nm was measured in the absence or presence of 1 μg/mL oligomycin (complex V inhibitor).
Measurement of mitochondrial membrane potentialMitochondrial membrane potential was evaluated using a previously described method (Sato et al., 2021).
Statistical analysesAll data are presented as the mean ± standard deviation (S.D.) unless otherwise specified. GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses. Statistically significant differences between conditions were determined using an unpaired Student's t-test for two groups or one/two-way analysis of variance (ANOVA) followed by Tukey’s tests. A statistical p-value < 0.05 was considered as statistically significant.
Our previous report suggests that LPS administration increases MPT pore opening susceptibility in rats (Arakawa et al., 2019); therefore, we investigated whether LPS administration would also increase MPT pore opening susceptibility in mice. Ca2+ treatment significantly induced swelling in the LPS-treated mitochondria compared to that of the vehicle-treated mitochondria and was inhibited by CsA (Figs. 1A and B). These results suggest that the enhancement of MPT pore opening susceptibility after LPS administration also occurs in mice. Our previous report suggests that heparin pretreatment reduces MPT pore opening susceptibility by LPS (Arakawa et al., 2019). However, the present study in mice showed that heparin pretreatment did not affect MPT pore opening susceptibility by LPS (Figs. 1C and D). The effects of LPS dose and liver isolation time after LPS administration on MPT pore opening susceptibility were also examined. The results showed that even low doses LPS (0.05 and 0.5 mg/kg) increased MPT pore opening susceptibility (Supplement Fig. 1A). However, shortening the time between LPS administration and liver isolation (≤ 1 hr) attenuated the effect (Supplement Fig. 1B). In addition, Ca2+-independent swelling was observed in the absence of Ca2+ in LPS-treated mitochondria. Ca2+-dependent swelling was enhanced by LPS, which was not completely inhibited by CsA (Figs. 1A and B). Interestingly, CsA-insensitive swelling was not observed in rats (Arakawa et al., 2019); therefore, this phenomenon seems to be mouse-specific.
Evaluation of mitochondrial swelling following LPS administration in the absence (A and B) or presence (C and D) of heparin in mouse liver mitochondria. Mitochondrial swelling was determined by incubating liver mitochondria in the absence or presence of 12.5 μM Ca2+ (MPT inducer) and 2 μM CsA (MPT inhibitor). Absorbance at 540 nm was monitored time-dependently (A and C) and at 528 sec (B and D). ΔAbs was defined by subtracting the absorbance at time 0. Data are shown as the mean ± S.D. (n = 3), **p < 0.01, ***p < 0.001, using two-way ANOVA analysis followed by Tukey test. N.S. = not significant.
Next, we examined the mechanism of MPT pore opening susceptibility enhancement in mice based on our previous reports (Arakawa et al., 2019). Oxidative stress was not affected by LPS administration in the liver mitochondria, whereas it was significantly increased in the liver homogenates (Fig. 2). Moreover, contrary to our previous findings in rats (Arakawa et al., 2019), LPS administration in mice resulted in a significant decrease in respiratory activity via complexes I and II (Fig. 3A) and its decrease was not counteracted by heparin treatment (Fig. 3B). Even low doses of LPS inhibited respiratory activity after the addition of ADP. Again, shortening the time between LPS administration and liver isolation (≤ 1 hr) attenuated the inhibitory effect on respiration (Supplement Figs. 2A and B). Based on this, we hypothesized that the function of the respiratory chain complex was disrupted by LPS administration in mice. Therefore, we evaluated the activity of respiratory chain complexes I to V. The activity of all respiratory chain complexes was not changed by LPS administration (Fig. 4). Based on these results, the mechanism of LPS-induced enhancement of MPT pore opening susceptibility differs between rats and mice.
Evaluation of oxidative stress by TBARS assay in mouse liver mitochondria and liver homogenate. Malondialdehyde (MDA; lipid peroxidation products) content was calculated as an indicator of oxidative stress. Data are shown as the mean ± S.D. (n = 5), *p < 0.05, using an unpaired Student’s t-test.
Evaluation of respiratory activity by measuring oxygen consumption using mouse liver mitochondria with LPS administration in the absence (A) or presence (B) of heparin. Glutamate and malate (complex I substrate) or succinate (complex II substrate) were added to mitochondria. After this, ADP was used as a substrate for oxidative phosphorylation. Time-dependent oxygen consumption via complex I or complex II was measured. Data are shown as the mean (n = 3).
Evaluation of respiratory chain complex activity using mouse liver mitochondria. Isolated mitochondria were frozen and thawed several times, and complex activity was determined from the change in absorbance by adding the substrate of each respiratory chain complex. Data are shown as the mean ± S.D. (n = 3) using an unpaired Student’s t-test. N.S. = not significant.
We focused on ANT function in the context of MPT pore opening susceptibility as (i) the decrease in respiratory activity following the addition of ADP was particularly remarkable (Fig. 3A), and (ii) ANT is a component of the MPT pore (Carrer et al., 2021; Bonora et al., 2022). We hypothesized that ANT-mediated ADP transport may be downregulated in LPS-treated mitochondria and thus evaluated respiratory activity by exposing the vehicle-treated mitochondria to carboxyatractyloside (CATR), a specific inhibitor of ANT. CATR treatment significantly decreased respiratory activity following the addition of ADP (Fig. 5), which was similar to the respiratory activity observed in LPS-treated mitochondria (Fig. 3). This suggests that ANT dysfunction may be involved in mitochondrial respiratory deficiency in LPS-treated mitochondria.
Evaluation of respiratory activity by measuring oxygen consumption in mouse liver mitochondria in the absence or presence of 2 μM CATR (ANT inhibitor). Glutamate and malate (complex I substrate) or succinate (complex II substrate) were added to mitochondria. After that, ADP was used as a substrate for oxidative phosphorylation. Time-dependent oxygen consumption via complex I (A) and complex II (B) was measured. Data are shown as the mean (n = 3).
ANT has two conformations for ADP/ATP exchange: the c-state (nucleotide-binding site facing the cytosol) and the m-state (nucleotide-binding site facing the mitochondrial matrix). The c-state enhances MPT pore opening susceptibility, whereas the m-state inhibits MPT pore opening induction (Brustovetsky, 2020). Based on this, we hypothesized that LPS administration in mice causes ANT to favour the c-state conformation, and thus results in increased MPT pore opening susceptibility. Therefore, we evaluated the effect of CATR, which binds to ANT in the c-state and fixes its conformation, on MPT pore opening susceptibility. Although CATR-induced swelling was observed in both groups, swelling was observed earlier in LPS-treated mitochondria than in vehicle-treated mitochondria (Fig. 6A). In addition, CsA-insensitive swelling was observed only after CATR treatment in LPS-treated mitochondria (Fig. 6B). Moreover, administration of bongkrekic acid, which binds to ANT in the m-state and fixes its conformation, partially suppressed the CsA-insensitive swelling observed in LPS-treated mitochondria (Supplement Fig. 3). This suggests that altered ANT conformation may contribute to enhanced MPT pore opening susceptibility in LPS-treated mitochondria.
Evaluation of mitochondrial swelling in mouse liver mitochondria by CATR treatment with CsA. Mitochondria were incubated in the absence (A) or presence (B) of 2 μM CsA. ΔAbs at 540 nm was monitored time-dependently. Data are shown as the mean ± S.D. (n = 3).
Our previous study suggested that LPS administration increases MPT pore opening susceptibility in rats by altering mitochondrial function (Arakawa et al., 2019); however, the mechanism by which MPT pore opening susceptibility is enhanced in mice is still unknown. Our results from the present study suggest that LPS administration increases MPT pore opening susceptibility (Figs. 1A and B) but decreases respiratory activity (Fig. 3A). Due to the nature of these results, we focused on ANT; respiratory activity following CATR treatment (an ANT inhibitor) was similar to that following LPS administration (Fig. 5). Additionally, CATR treatment increased MPT pore opening susceptibility in LPS-treated mitochondria compared to that of vehicle-treated mitochondria (Fig. 6). These results suggest that ANT was in the c-state upon LPS administration and that its conformation contributed to increased MPT pore opening susceptibility in mouse mitochondria.
Our previous study also suggested that mitochondrial oxidative stress increases MPT pore opening susceptibility in rats (Arakawa et al., 2019); however, this had not been confirmed in mice (Fig. 2). We hypothesized that the alteration of ANT function affects the generation of oxidative stress. ANT functions not only as adenine nucleotide transporter, but also as uncoupling proteins. The uncoupling function (that is, the mitochondrial proton leak function) is inhibited during ADP/ATP transport (Bertholet et al., 2019). In addition, uncoupling-related proton leak inhibits the generation of oxidative stress (Cadenas, 2018). The present study confirmed that the mitochondrial membrane potential in the LPS-treated mitochondria was significantly decreased (Supplement Fig. 4).
While we did not examine the mechanism of alteration of ANT conformation, we believe that cardiolipin may be involved. Cardiolipin, a mitochondrial-specific phospholipid (Segawa et al., 2018), maintains and regulates mitochondrial structure and function, respectively. Indeed, cardiolipin binds to ANT (Beyer and Klingenberg, 1985) which allows for the regulation of ADP/ATP transport (Hoffmann et al., 1994). Cardiolipin is broken down into monolysocardiolipin and free fatty acid by phospholipase A2. The binding of monolysocardiolipin to ANT is weak, thus ANT function is reduced (Hoffmann et al., 1994; Beyer and Nuscher, 1996; Imai et al., 2003). In addition, free fatty acids are known to interact with ANT, stabilizing it as a conformation of c-state, and increasing MPT pore opening susceptibility (Schönfeld and Bohnensack, 1997). Phospholipase A2 is activated by decreased mitochondrial membrane potential (Rauckhorst et al., 2014). The present study showed that there is a decrease in mitochondrial membrane potential following LPS administration (Supplement Fig. 4). We believe that cardiolipin decomposition contributes to decreased ANT activity and stabilization of its c-state conformation. For cardiolipin, MPT pore opening susceptibility was involved with not only monolysocardiolipin formation but also cardiolipin hydroperoxide formation (Segawa et al., 2018). Indeed, our previous study in rat suggests that LPS treatment increases cardiolipin hydroperoxide level (Arakawa et al., 2019). In any case, cardiolipin is likely to be involved in the opening of the MPT pore by LPS at this time, but the mechanism differs between rats and mice.
Our results in relation to respiratory activity (Fig. 3A) were similar to those of liver-specific ANT1/2 knockout-derived liver mitochondria (Kokoszka et al., 2004). In contrast, ANT1/2 knockout is non-lethal (Kokoszka et al., 2004) and does not necessarily denote AMPK phosphorylation, which is generally activated by decreasing ATP levels (Cho et al., 2017). Based on these studies, when ADP cannot be transported via ANT in vivo, an alternative pathway is activated in order to produce energy. Additionally, lactate levels remain unchanged in ANT1/2 knockout mice (Cho et al., 2017). Energy metabolism therefore does not shift from oxidative phosphorylation to glycolysis; ADP is instead transported by an alternative pathway.
Our study suggested that CsA-insensitive swelling was not observed in vehicle-treated mitochondria following CATR treatment (Fig. 6B). Considering the various functions of LPS (Alexander and Rietschel, 2001), we believe that other factors may be involved in MPT pore opening susceptibility. García reported that CsA-insensitive swelling is not induced by CATR treatment in mitochondria immediately after isolation, but is observed in mitochondria under conditions of stress (García et al., 2009). LPS administration may have caused structural changes in ANT conformation and stress in mitochondria. Based on these, we believe that vehicle-treated mitochondria were stress-free condition; therefore, CsA-insensitive swelling was not observed by CATR treatment.
We could not elucidate the mechanism underlying the inhibition of respiratory activity following LPS administration. We confirmed that respiratory activity was not inhibited in primary mouse hepatocytes after LPS treatment (Supplement Fig. 5). On the other hand, the increase in MPT pore opening susceptibility, CsA-insensitive swelling, and inhibition of respiratory activity were diminished in C3H/HeJ (TLR-4 mutation mice strain (Poltorak et al., 1998)) compared with those in C3H/HeN mice (Supplement Figs. 6 and 7). These results suggest that TLR-4 signaling is certainly a major factor for enhancing MPT pore opening susceptibility, but it is unlikely to be the result of the direct action of LPS on hepatocyte. We attempted to determine which cells are involved in respiratory inhibition, using precision-cut liver slices (PCLS) and flow cytometry. After preparing PCLS in control mice, LPS treatment did not increase lactate level in the supernatant (assuming that respiratory inhibition results in increased lactate production; Supplement Fig. 8A). On the other hand, lactate level was increased in PCLS prepared after in vivo administration of LPS (Supplement Fig. 8B). Generally, PCLS prepared from control mice contained Kupffer cells (liver-resident macrophages) but not (very few, if any) monocyte-derived macrophages. Furthermore, after 2 hr of LPS treatment, monocyte-derived macrophage, but not neutrophil or Kupffer cells, accumulated in the liver (Supplement Figs. 8C–F). These results suggest that monocyte-derived macrophages may be involved in respiratory inhibition and subsequent enhancement of MPT pore opening susceptibility. In the future, it is also necessary to investigate how macrophages affect mitochondrial function in hepatocytes.
In summary, MPT pore opening susceptibility is increased following LPS administration in mice by a different mechanism from that in rats. In mice, the alteration of ANT conformation by LPS treatment contributes to MPT pore opening susceptibility. We propose that more studies are needed on this topic, particularly considering that the mechanism of changes in MPT pore opening susceptibility may vary among species. This is especially relevant when extrapolating animal study results to humans.
This work was supported by AMED (grant number JP21fk0310112 [K.I.]). We would like to thank Editage (www.editage.com) for the English language editing.
Conflict of interestThe authors declare that there is no conflict of interest.
